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Construction method of aptamer sensor for measuring ochratoxin A

A technology of aptamer sensor and ochratoxin, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of difficult effective preservation of clones, high production costs, and long antibody preparation cycles, etc. question

Active Publication Date: 2013-02-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using ochratoxin A aptamers instead of antibodies as ochratoxin A recognition probes, its in vitro screening and in vitro preparation characteristics can effectively overcome the long antibody preparation cycle, high production costs, technical difficulties, large batch-to-batch differences, and cloning problems. Strains (cells) are difficult to effectively preserve the disadvantages

Method used

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  • Construction method of aptamer sensor for measuring ochratoxin A
  • Construction method of aptamer sensor for measuring ochratoxin A
  • Construction method of aptamer sensor for measuring ochratoxin A

Examples

Experimental program
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Embodiment 1

[0027] (1) Streptavidin-coated PCR tube

[0028] First, coat the PCR tube with 50 μL of 0.8% glutaraldehyde solution at 37°C for 5 hours, then wash the PCR tube three times with ultrapure water for 3 minutes each time; Coat PCR tubes with 12.5ng / mL streptavidin, incubate 30 μL of each tube at 37°C for 2 hours, and then wash with the above carbonate buffer three times, each time for 3 minutes, to remove unbound streptavidin and prime;

[0029] (2) The aptamer hybridizes with its partially complementary DNA fragment and immobilizes on the surface of the PCR tube

[0030] with 750mM NaCl, 75 mM C 6 h 5 Na 3 o 7 , pH 8.0 hybridization buffer to dilute the aptamer and part of the DNA fragments complementary to the aptamer to 50nM and 100nM respectively, and mix the two thoroughly at a volume ratio of 1:1, and add the mixture to each PCR tube 30 μL, and incubate at 37°C for 40 minutes to allow the complementary double strands to fully hybridize and bind to the surface of the P...

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Abstract

The invention discloses a construction method of an aptamer sensor for measuring ochratoxin A, belonging to the technical field of detection of a biosensor. The construction method comprises the following steps of coating a PCR (Polymerase Chain Reaction) tube by streptavidin, hybridizing an aptamer and partially complementary DNA (Deoxyribose Nucleic Acid) fragments and fixing on the surface of the PCR tube, and constructing the aptamer sensor. The invention provides a single stranded DNA aptamer combined with the ochratoxin A at high specificity and high affinity, and DNA fragments, which are partially complementary to the aptamer, through recognition combination of the aptamer and the ochratoxin A, aptamer conformation is induced to change so as to release the DNA fragments, which are partially complementary to the aptamer, the fragments, which are partially complementary to the aptamer, are taken as PCR amplification templates, and the ochratoxin A is detected through amplified fluorescence signals. According to the method provided by the invention, ultra-sensitivity detection of the ochratoxin A is realized at low detection limit, high detection sensitivity and good specificity, and an effective method is provided for detection of the trace ochratoxin A and the other harmful substances.

Description

technical field [0001] The invention discloses a method for constructing an aptamer sensor for measuring ochratoxin A, which belongs to the technical field of biosensor detection. Background technique [0002] Ochratoxins (Ochratoxins) are a group of structurally similar toxic metabolites produced by several species of Aspergillus and Penicillium. There are four compounds A, B, C, and D, including 7 compounds with similar chemical structures, of which Ochratoxin A (OTA) is the most toxic, the most widely distributed, the highest toxin production, the most serious pollution to crops, and the most closely related to human health. The main producing bacteria are Asperillus ochraceus and Penicillium verrucosum, which have a wide range of food contamination and widely exist in various foods and feeds such as grains, coffee beans, beans, raisins, beer, and grape juice. Among them, grains and their by-products are the main source of OTA. Ochratoxin A has multiple toxicities such ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 胥传来徐丽广匡华马伟刘丽强宋珊珊
Owner JIANGNAN UNIV
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